Wroble BN , Finkielstein CV , Sible JC .

GM076112 NIGMS NIH HHS , GM59688 NIGMS NIH HHS , R01 GM059688 NIGMS NIH HHS , R01 GM076112 NIGMS NIH HHS

	Figure 1. Overexpression of Wee1 lengthens pre-MBT cell cycles. One-cell stage embryos were microinjected with 2.5 ng mRNA encoding Wee1 or luciferase (control). (A) Embryos were collected at indicated times post-fertilization (hr pf) and analyzed for Wee1 protein by Western analysis. (B) Xenopus embryos injected with 2.5 ng Wee1 or luciferase mRNA were photographed at 4.5 hr pf, when embryos expressing exogenous Wee1 were delayed approximately one cell cycle compared to luciferase controls. (C) Overexpression of Wee1 results in premature tyrosine phosphorylation of Cdks. Embryo lysates were subjected to Western analysis using a phosphoCdk antibody (Cell Signaling Technology). Both phosphorylated p34Cdk1 and p32Cdk2, are recognized by this antibody, which likely accounts for the bands of slower and faster mobility, respectively. Phosphorylation of Cdks was apparent as early as Stage 6 (3 hrs pf) in embryos injected with Wee1. (D) Embryos (n = 10) were collected at the indicated stages, then DNA was isolated and resolved by agarose gel electrophoresis. Approximate timing of the MBT in luciferase expressing control embryos is indicated. E) Embryos were collected at Stage 9 (7 hrs pf), fixed, sectioned, stained with DAPI to visualize nuclear morphology, and photographed. A representative field is shown. scale bar = 50 μm
	Figure 2. Overexpression of Wee1 in early Xenopus embryos triggers apoptosis after the MBT. One-cell stage Xenopus embryos were microinjected with 2.5 ng wild-type (WT) Wee1, kinase-dead (KD) Wee1, or luciferase (control) mRNA. (A-B) Embryos expressing Wee1, KDWee1, and luciferase developed normally until the early gastrula stage, when embryos expressing exogenous WT Wee1 exhibited abnormal morphology consistent with apoptosis. Embryos shown were photographed at gastrulation (Stage 11). (C-D) Embryo extracts were collected at Stage 11 and incubated with recombinant human PARP, a substrate for caspase 3 [4]. The presence of a cleaved PARP fragment indicates caspase activity, a marker of apoptosis.
	Figure 3. Overexpression of Wee1 delays the MBT and alters Cdk2. Embryos were injected at the one-cell stage with 2.5 ng of Wee1 or luciferase (control) mRNA. (A, B) Embryos were collected at the indicated stages and lysates were subjected to Western analysis using (A) Cdc25A and (B) cyclin E antibodies. Approximate timing of the MBT in luciferase expressing control embryos is indicated. (C) Embryos were collected at 4 hrs pf and lysates were immunoprecipitated with cyclin E serum. Immunoprecipitates were subjected to Western analysis using a primary phosphoCdk antibody (Cell Signaling Technology). (D) Cyclin E immunoprecipitates were collected at the times indicated and assayed for Cdk2 activity by phosphoryation of histone H1. The amount of γ-32P ATP incorporated in histone H1 is expressed as radioactive counts per minute (CPM).
	Figure 4. Inhibition of Cdk2 triggers apoptosis. One-cell stage embryos were injected with 5 ng of δ34-Xic1 or p27Xic1CK- (control) protein. (A) Embryos expressing δ34-Xic1 were delayed approximately one cell cycle at Stage 8 (5 hr pf) compared to p27Xic1CK- controls. At Stage 11 (12 hr pf), embryos expressing δ34-Xic1 exhibited abnormal morphology consistent with apoptosis compared to p27Xic1CK- controls, which gastrulated. p27Xic1CK- controls continued to develop through gastrulation and formed a neural groove, evident at Stage 13 (16 hr pf). (B) Extracts of Stage 11 embryos were incubated with recombinant PARP, a substrate for caspase 3 [4]. The presence of a cleaved PARP fragment indicates caspase activity. Replicate samples are shown.
	Figure 5. Effects of inhibitors of Cdk activity on early development of X. laevis. (A) Exogenous Chk1/2 indirectly inhibits Cdk1 and Cdk2 by targeting Cdc25A for degradation. Onset of the MBT is delayed, but development proceeds without apoptosis. (B) Exogenous Wee1 phosphorylates Cdk1 and Cdk2. The total pool of Cdk1 activity is inhibited. Onset of the MBT including degradation of Cdc25A is delayed. Embryos die by apoptosis during early gastrulation. (C) Exogenous δ34-Xic1 inhibits Cdk2 but not Cdk1. Like Wee1, δ34-Xic1 delays the MBT including degradation of Cdc25A, and induces apoptosis.

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